SUPPLEMETARY MATERIAL for

Tumor-Penetrating iRGD Peptide Inhibits Metastasis

Kazuki N. Sugahara1,2, Gary B. Braun1,3, Tatiana Hurtado de Mendoza1, Venkata Ramana Kotamraju1, RandallP. French4, Andrew M. Lowy4, Tambet Teesalu1,5and Erkki Ruoslahti1,3

1Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.

2Department of Surgery, Columbia University College of Physicians and Surgeons, New York, New York, USA.

3Center for Nanomedicine and Department of Cell, Molecular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, USA.

4Division of Surgical Oncology and Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.

5Centre of Excellence for Translational Medicine, University of Tartu, Tartu, Estonia.

Running Title: iRGD inhibits metastasis and repels tumor cells

Key Words:tumor-penetrating peptides, C-end Rule, neuropilin, metastasis, chemorepulsion

Corresponding Author:

Kazuki N. Sugahara, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858-646-3100 x3517; Fax: 858-795-5353; E-mail: .

List of Abbreviations:VEGF, vascular endothelial growth factor; NRP-1, neuropilin-1; NRP-2, neuropilin-2; CendR, C-end Rule; iRGD, a cyclic peptide with an amino acid sequence of CRGDK/RGPD/EC (the CRGDKGPDC form was used in the current study); CRGDK/R, a linear penta-peptide; PDAC, pancreatic ductal adenocarcinoma; KPC mice, mice with KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mutations; DMEM, Dulbecco’s Modified Eagle Medium; FBS, fetal bovine serum; BSA, bovine serum albumin; iNGR, a cyclic peptide with an amino acid sequence of CRNGRGPDC; PFA, paraformaldehyde; AgNPs, silver nanoparticles; NA, NeutrAvidin; PEG, polyethylene glycol; OPSS, orthopyridyl disulfide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA, analysis of variance; RGDfV, a cyclic peptide; iRGDD, a scrambled iRGD peptide with an amino acid sequence of CRGDDGPKC; CRGDC, a cyclic peptide; iRGD-AgNPs, iRGD-coated AgNPs; CRGDC-AgNPs, CRGDC-coated AgNPs; Sema3A, semaphorin 3A; Sema3F, semaphorin 3F; RPARPAR, a prototypic linear CendR peptide; RPARPAR-NH2, RPARPAR peptide with a blocked C terminus; CRGEC, a control peptide for CRGDC.

Financial Support: This work was supported by grants R01CA167174 (K.N. Sugahara) and R01CA152327 (E.Ruoslahti) from the National Cancer Institute of NIH, Career Development Award from American Association of Cancer Research/Pancreatic Cancer Action Network (K.N.Sugahara), and Blasker Science Award from the San Diego Foundation (K.N.Sugahara). G.B.Braunwas supported by CA121949 NIH T32 Fellowship, and T.Teesaluwas supported by KG110704 Susan Komen for Cure Foundation Career Development Award, European Research Council Starting Grant (GliomaDDS), and Wellcome Trust International Fellowship (WT095077MA).

Conflict of Interest: K.N. Sugahara, V.R. Kotamraju, T. Teesalu, and E. Ruoslahti have ownership interest (including patents) in CendR Therapeutics Inc. E. Ruoslahti is also founder, chairman of the board, consultant/advisory board member of CendR Therapeutics Inc. No potential conflicts of interest were disclosed by the other authors.

Supplementary Figure:7

Supplementary Table:1

Supplementary Movie:5

SupplementaryFigureS1.Correlation betweenmetastatic burden and primary tumor weight after long termin vivopeptide treatment. A, Metastatic burden and primary tumor weight after iRGD treatment in GFP-PC-3 tumor mice (refer to Fig. 1C and D)were subjected to regression analysis using Prism software. Pearson’s test was performed to calculate correlation coefficient.B, iRGD and iNGR data sets obtained in the LM-PmC tumor treatment studies (refer to Fig. 3B and C)were analyzed as described above.

Supplementary Figure S2. Targeting of LM-PmCpancreatic tumor cells by iRGD and iNGR. A,Neuropilin and v integrin expression in LM-PmC mouse pancreatic cancer cells analyzed by flow cytometry. The profiles represent the values of cells incubated with isotype control (red) or appropriate neuropilin (NRP) or integrin antibodies (blue) as primary antibodies. B, NRP-1-dependent in vitro penetration of fluorescamine (FAM)-conjugated iRGD and iNGR (CRNGRGPDC) into LM-PmC cells (red). Green, peptides; blue, nuclei. Scale bars = 20 m. C, Confocal micrographs of orthotopic LM-PmC tumors harvested from mice injected with FAM-iRGD or FAM-iNGR. Red: LM-PmC cells; green: peptides; blue: nuclei.Squared areas are magnified in the insets. Note that both FAM-iRGD and FAM-iNGR target individual LM-PmC cells even at the invasion front. Representative fields from multiple sections of three tumors are shown. Scale bars = 200 m.

SupplementaryFigureS3. Effects of RGD peptides on tumor cell attachment to vitronectin. LM-PmC (A) or GFP-PC-3 (B) cells were allowed to attach to 96-well plates coated with vitronectin in the presence of various concentrations of iRGD, CRGDC, or RGDfV. Attached cells were quantified using MTT assays.n = 3. Error bars denote mean ± SEM.

SupplementaryFigureS4.In vitro tumor cell treatment with CendR peptides. LM-PmC (A) or GFP-PC-3 (B) cells cultured on collagen-I-coated surfaces in fully supplemented DMEM for 24 hours were treated with increasing amounts of iRGD, iNGR, or RPARPARfor 48 hours in a 37˚C CO2 incubator. Live cells were quantified with MTT (n = 3). Error bars denote mean ± SEM.

SupplementaryFigureS5.Design of chemorepulsion assays. Silver nanoparticles coated with iRGD (iRGD-AgNPs) or CRGDC (CRGDC-AgNPs), or plain AgNPs were immobilized as round spots on glass bottom wells. Tumor cells at a concentration of 104 cells in 100 l in fully suppliedDMEM medium were seeded in the center of the immobilized AgNPs using cloning cylinders, and were allowed to attach to the surface for 3 hours. The cloning cylinders were removed, and the cells were incubated in culture medium containing 1% BSA. The wells were sujected to live cell imaging for 48 hours with an Inverted Olympus IX81 Wide Field and Fluorescence Microscope equipped with CO2 and Temperature Controlled Time Lapse System.

SupplementaryFigureS6.iRGD inhibits GFP-PC-3 cell attachment to fibronectin in a NRP-1-dependent manner.A and B, Cell attachment assays. The number of GFP-PC-3 cells that attached to fibronectin-coated wells in the presence of various peptides was quantified. In A,iRGD, CRGDK, iNGR, RPARPAR, and RPARPAR-NH2 were used. In B, CRGDC and CRGEC were used. Some cells were also treated with anti-NRP-1 b1b2 or control IgG. n = 3 per experiment. Non-treated columns were considered as 100%.Error bars, mean ± SEM; statistical analyses, ANOVA; n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Statistics against non-treated columns are shown unless otherwise noted.C,Cell retraction assays.GFP-PC-3 cells (green)cultured on fibronectin-coated coverslips were treated with 10 M of the indicated peptides for 1 hour, fixed, and stained for phospho-paxillin (red) and nuclei (blue). Representative confocal micrographs from three independent experiments are shown. Scale bars = 20 m.

SupplementaryFigureS7. CendR peptides do not affect tumor cell attachment to collagen type-I. LM-PmC (closed bars) or GFP-PC-3 (open bars) cells were treated with iRGD, CRGDK, iNGR, RPARPAR or RPARPAR-NH2 for 30 minutes at 37˚C under mild rotation. The cells were then seeded into 96-well plates coated with collagen type-I, and allowed to attach for 30 minutes at 37˚C in the presence of the peptides. The number of cells that remained attached to the wells were quantified using MTT.Cell attachment without any peptide was considered as 100%. n = 3. Error bars denote mean ± SEM.

Supplementary Movie SM1. Effects of iRGD on tumor cell motility analyzed by live cell imaging. LM-PmC cells were seeded on glass bottom slides in close proximity to iRGD-coated silver nanoparticles (iRGD-AgNPs). Microscopic time-lapse images were taken every 15 minutes for 48 hours. The dark area in the right upper corner is coated with iRGD-AgNPs. Note that the cells are repelled by iRGD. A representative video from five independent experiments is shown.

Supplementary Movie SM2. Cell retraction in response to iRGD analyzed by live cell imaging. LM-PmC cells were seeded on glass bottom slides in close proximity to iRGD-coated silver nanoparticles (iRGD-AgNPs). Microscopic time-lapse images were taken every 15 minutes for 48 hours. The dark area in the right lower corner is coated with iRGD-AgNPs. Note the dramatic collapse of the cellular processes after they interact with the iRGD-AgNPs. A representative video from five independent experiments is shown.

Supplementary Movie SM3. Alteration of cell motility in response to iRGD analyzed by live cell imaging. LM-PmC cells were seeded on glass bottom slides in close proximity to iRGD-coated silver nanoparticles (iRGD-AgNPs). Microscopic time-lapse images were taken every 15 minutes for 48 hours. The dark area in the right lower corner is coated with iRGD-AgNPs. Note that the highly motile cell, which moves to the center, does not migrate beyond the boarder line of iRGD-AgNPs. The cells on the right that landed on top of the particles collapse, while the cells on the left remain spread on theglass surface. A representative video from five independent experiments is shown.

Supplementary Movie SM4. Effects of CRGDC on tumor cell motility analyzed by live cell imaging. LM-PmC cells were seeded on glass bottom slides in close proximity to CRGDC-coated silver nanoparticles (CRGDC-AgNPs). Microscopic time-lapse images were taken every 15 minutes for 48 hours. The dark area on the right side is coated with CRGDC-AgNPs. Note that the cells progressively migrate over the CRGDC-AgNPs and completely saturate the field in 48 hours. A representative video from five independent experiments is shown.

Supplementary Movie SM5. Effects of plain silver nanoparticles on tumor cell motility analyzed by live cell imaging. LM-PmC cells were seeded on glass bottom slides in close proximity to plain silver nanoparticles (AgNPs). Microscopic time-lapse images were taken every 15 minutes for 48 hours. The dark area in the right is coated with the AgNPs. Note that the cells randomly migrate on the surface regardless of the presence of AgNPs and completely saturate the field in 48 hours. A representative video from five independent experiments is shown.

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